Calcium ions (Ca2+) are one of the most ubiquitously used signaling molecules in eukaryotic cell regulation. They regulate a range of cellular processes such as muscle contraction, hormone secretion or gene transcription. Ca2+ enters the cells via multiple Ca2+ entry routes formed by Ca2+ channels and transporters and is very efficiently eliminated by Ca2+ pumps and exchangers. The tight control of the cells Ca2+ homeostasis is essential for cellular signaling and for the maintenance of cellular integrity. Recent studies identified two protein families (STIM and Orai/CRACM) that mediate a specific form of Ca2+ entry, termed store-operated calcium entry (SOCE). STIM1, is an endoplasmatic reticulum (ER) resident protein that rapidly translocates to the plasma membrane (PM)-adjacent compartment of the ER upon depletion of the ER Ca2+ stores where it activates the calcium channel, Orai1. The importance of this calcium entry pathway is highlighted by the fact that mutations in Orai1 has been linked to severe inborn human immunodeficiencies and that this route of calcium entry is key to the calcium regulated activation of T-cells mediated by the NFAT transcription factors. Studying the molecular details of STIM1 translocation to the PM-adjacent ER compartment and identification of the domains responsible for STIM1/Orai1 interaction will help develop novel molecular approaches for immunosupression. As most ion channels and transporters are regulated by the minor but critically important class of acidic phospholipids, the phosphoinositides, the phosphoinositide dependence of Orai1 channel activation in the PM and of STIM1 movements from the tubular to PM-adjacent ER regions during Ca2+ store depletion were investigated. Phosphatidylinositol 4,5-bisphosphate PtdIns(4,5)P2 levels were changed either with agonist stimulation, or by chemically-induced recruitment of a phosphoinositide 5-phosphatase domain to the PM, while PtdIns4P levels were decreased by inhibition or down regulation of phosphatidylinositol 4-kinases (PI4Ks). Agonist-induced phospholipase C activation and PI4K inhibition but not isolated PtdIns(4,5)P2 depletion substantially reduced endogenous or STIM1/Orai1 mediated SOCE without preventing STIM1 movements toward the PM upon Ca2+ store depletion. Patch clamp analysis of cells overexpressing STIM1 and Orai1 proteins confirmed that phospholipase C activation or PI4K inhibition greatly reduced ICRAC currents (the electrophysiological correlate of STIM1/Orai1 mediated SOCE). These results suggest an inositide requirement of Orai1 activation but not STIM1 movements and indicate that PtdIns4P rather than PtdIns(4,5)P2 is a likely determinant of the Orai1 channel activity. In another set of studies it was examined whether subplasmalemmal mitochondria are located in close proximity to the ER-PM regions close to the activated Orai1 channels. This question was important as such a group of mitochondria could preferentially respond to Ca2+ influx occuring via the STIM1/Orai1 mechanism. For this purpose COS-7 cells were cotransfected with Orai1, STIM1 labeled with YFP or mRFP and a mitochondrially targeted Ca2+ sensitive fluorescent protein, inverse Pericam. Depletion of ER Ca2+ with ATP + thapsigargin (Tg) (in Ca2+ -free medium) induced the appearance of STIM1 puncta in the &#8804;100 nm wide subplasmalemmal space, as examined with total internal reflection fluorescence (TIRF) microspcopy. In such cells, mitochondria were located either in the gaps between STIM1-tagged puncta or in remote, STIM1-free regions. After addition of Ca2+ to initiate Ca2+ influx via the activated Orai1 channles, mitochondrial (Ca2+m) increased similarly regardless of the mitochondrion-STIM1 distance. These observations indicated that specially positioned mitochondria are not likely to serve as uniquely sensitive sensors of Ca2+ influx occuring at the PM and the subplasmalemmal ER. Another research focus of the group was the analysis of the phosphoinositide changes upon invasion of mammalian cells by the enteropathogenic bacteria, E. coli (EPEC). EPEC is a major cause of severe infantile diarrhea in developing countries. Many bacterial pathogens use the cells own trafficking machinery to invade and move around within the cells and help spred the bacteria from one cell to another. Understanding the underlying molecular events will help us find new strategies to better fight bacterial infections. Phosphatidylinositol 4,5-bisphosphate PtdIns(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate PtdIns(3,4,5)P3 are phosphoinositides (PIs) present in small amounts in the inner leaflet of the plasma membrane (PM) lipid bilayer of host target cells. They modulate the activity of proteins involved in EPEC infection. However, the role of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in EPEC pathogenesis remains obscure. In a set of experiments utilizing the fluorescent phosphoinositide probes developed in our laboratory, we collaborated with Dr. Benjamin Aroetis group to show that EPEC induces a transient PtdIns(4,5)P2 accumulation at bacterial infection sites. Simultaneous actin accumulation, likely involved in the construction of the actin-rich pedestal, was also observed at these sites. Acute PtdIns(4,5)P2 depletion partially diminished EPEC adherence to the cell surface and actin pedestal formation. These findings were consistent with a bimodal role, whereby PtdIns(4,5)P2 contributes to EPEC association with the cell surface and to the maximal induction of actin pedestals. Finally, it was shown that EPEC induces PtdIns(3,4,5)P3 clustering at bacterial infection sites, in a translocated intimin receptor (Tir)-dependent manner. Tir phosphorylated on tyrosine 454, but not on tyrosine 474, formed complexes with an active phosphatidylinositol 3-kinase (PI3K), suggesting that PI3K recruited by Tir prompts the production of PtdIns(3,4,5)P3 beneath EPEC attachment sites. The functional significance of this event may be related to the ability of EPEC to modulate cell death and innate immunity.